An Atomic Model of the
Interferon-b Enhanceosome
Daniel Panne,1 Tom Maniatis,2 and Stephen C. Harrison1,*
1
The Jack and Eileen Connors Structural Biology Laboratory, Harvard Medical School, Department of Biological Chemistry and
Molecular Pharmacology, Howard Hughes Medical Institute, 250 Longwood Avenue, Boston, MA 02115, USA
2
Harvard University, Department of Molecular and Cellular Biology, 7 Divinity Avenue, Cambridge, MA 02138, USA
*Correspondence: harrison@crystal.harvard.edu
DOI 10.1016/j.cell.2007.05.019
SUMMARY
Transcriptional activation of the interferon-b
(IFN-b) gene requires assembly of an enhanceo-
some containing ATF-2/c-Jun, IRF-3/IRF-7, and
NFkB. These factors bind cooperatively to the
IFN-b enhancer and recruit coactivators and
chromatin-remodeling proteins to the IFN-b
promoter. We describe here a crystal structure
of the DNA-binding domains of IRF-3, IRF-7,
and NFkB, bound to one half of the enhancer,
and use a previously described structure of the
remaining half to assemble a complete picture
of enhanceosome architecture in the vicinity
of the DNA. Association of eight proteins with
the enhancer creates a continuous surface
for recognizing a composite DNA-binding ele-
ment. Paucity of local protein-protein contacts
suggests that cooperative occupancy of the
enhancer comes from both binding-induced
changes in DNA conformation and interactions
with additional components such as CBP. Con-
tacts with virtually every nucleotide pair account
for the evolutionary invariance of the enhancer
sequence.
INTRODUCTION
Transcriptional enhancers are cis-regulatory elements of
eukaryotic gene expression, often located at considerable
distances from the promoters they regulate (Carey and
Smale, 2000). Activation and nuclear localization of spe-
cific sets of transcription factors in response to specific
signals determine the occupancy of any particular en-
hancer, which can thus detect and integrate information
from multiple signal transduction pathways (Ptashne and
Gann, 2002). The multiple sites within enhancers create a
combinatorial code, which directs the transcriptional
machinery to specific promoters in response to a given
signal. Enhancers vary greatly in length and complexity. At
one end of the complexity gradient are enhancers with
transcription-factor-binding sites tightly clustered in a rela-
Cell 129, 1111–1123, June 15, 2007 ª2007 Elsevier Inc. 1111
tively compact genomic segment (e.g., the 55 base pairs
[bp] of the enhancer for the interferon-b [IFN-b] gene).
Transcription from promoters they regulate appears to
require cooperative formation of an ‘‘enhanceosome’’—an
assembly of distinct sets of proteins on the enhancer
DNA (Thanos and Maniatis, 1995). At the other end of
the gradient are ‘‘modular’’ enhancers, with rather loosely
clustered binding sites, generally covering much longer
stretches of the genome. Whereas enhanceosome as-
sembly ensures that enhancers operate as a single unit,
modular enhancers represent a more flexible form of
information processing (Arnosti and Kulkarni, 2005).
The best-characterized compact enhancer in the human
genome is the virus-inducible enhancer of the IFN-b gene.
Binding sites for the heterodimer ATF-2/c-Jun, interferon
response factors IRF-3 and IRF-7, and NFkB (p50:RelA)
are tightly clustered in a 55 bp stretch of DNA. The
‘‘AT-hook’’ protein HMGA1 (formerly designated HMGI(Y))
also binds to this sequence and promotes enhanceosome
assembly (Thanos et al., 1993), although the work reported
here shows that it is unlikely to be present in the final as-
sembly. The enhanceosome components bind to a nucleo-
some-free region of the IFN-b promoter, spanning the
interval from 102 to 47 bp relative to the transcription
start site. The IFN-b enhancer has been subdivided into
four positive regulatory domains (PRDs). The structure de-
scribed here shows that the enhancer forms essentially
one, composite binding element and that binding sites
overlap, but the PRD designations still remain useful. IRF
proteins bind to PRDI and III; NFkB to PRDII; and ATF-2/
c-Jun to PRDIV (Figure 1). Each of these factors can inter-
act with the coactivator, CREB-binding protein (CBP), or
with its closely related paralog, p300. Assembly of all these
factors into an ‘‘enhanceosome’’ is thought to provide
stringent specificity and stability (Maniatis et al., 1998;
Munshi et al., 1999). In vivo, the IFN-b enhancer is nucleo-
some free. It is flanked by two nucleosomes, one of which
masks the TATA box and the start site of transcription
(Agalioti et al., 2000). Virus infection leads to the activation
of ATF-2/c-Jun, IRF-3/7, and NFkB and their binding to the
nucleosome-free enhancer. Nucleosome acetylation and
chromatin remodeling by the SWI/SNF complex reposition
the nucleosome covering the TATA box and allow access
by TATA-binding protein (TBP) (Lomvardas and Thanos,
2001).

Figure 1. Strategy for Assembling the Complete INF-b Enhan-
ceosome Structure
Three sets of overlapping crystal structures were determined. (A) ATF-
2/c-Jun in complex with two IRF-3 molecules on the PRDIV and PRDIII
regions of the enhancer (Panne et al., 2004). (B) NFkB in complex with
IRF-7 and IRF-3 on PRDI–PRDII. (C) Four IRF-3 molecules in complex
with the full-length enhancer. (D) Fully assembled INF-b enhanceo-
some.
The IFN-b enhanceosome is an example of signal inte-
gration through assembly of a set of ‘‘generic’’ transcrip-
tion factors, each of which works in conjunction with other
factors at other enhancers. The individual factors do not
activate IFN-b gene expression by themselves, and failure
to mobilize any one of the factors abrogates IFN-b tran-
scription entirely (Maniatis et al., 1998). Although multiple
copies of individual binding sites can function as virus-
inducible elements, these artificial enhancers can respond
to stimuli other than virus infection (Thanos and Maniatis,
1995). The authentic enhanceosome is thus a coincidence
detector. It responds to the coordinate activation of a
specific set of transcription factors, which assemble on
the IFN-b enhancer into a complex for recruiting RNA
polymerase II.
The IRF family of transcription factors includes nine
mammalian members, IRF-1 to IRF-9, as well as several
viral homologs (Mamane et al., 1999). All IRFs are charac-
terized by a well-conserved 120 amino-acid N-terminal
DNA-binding domain, which recognizes similar DNA se-
quences termed IRF-binding element/IFN-stimulated re-
sponse element (ISRE), the consensus being 50 -AANNGA
AA-30 (Fujii et al., 1999). A challenge for structural studies
of INF-b regulation has been uncertainty concerning the
1112 Cell 129, 1111–1123, June 15, 2007 ª2007 Elsevier Inc.
occupancy of the four IRF-binding sites in the PRDIII-I re-
gion during different stages of viral infection and in differ-
ent cell types because techniques such as overexpression
by transient transfection frequently mask in vivo specific-
ity. Initial studies implicated IRF-1 in IFN-b transcription
(Fujita et al., 1988; Thanos and Maniatis, 1995), but later
gene-inactivation studies showed that IRF-1 is not re-
quired for viral induction of IFN-b in most cell types, and
that IRF-3 and IRF-7 are the relevant factors (Honda
et al., 2005; Sato et al., 2000; Wathelet et al., 1998). IRF-3
is constitutively expressed, but levels of IRF-7 are in-
creased through positive feedback by IFN-a/b stimulation
(Marie et al., 1998; Sato et al., 1998). These properties
suggested that IRF-7 has a role in later stages of virus in-
fection and that the immediate early enhanceosome might
contain only IRF-3 (Sato et al., 2000). We (Figure S1 avail-
able with this article online) and others (Escalante et al.,
2007) have therefore attempted to crystallize the enhan-
ceosome with IRF-3 bound to the PRDI-III region. It is
now clear, however, that IRF-7 is constitutively expressed
at high levels in plasmocytoid dendritic cells, the primary
source of type I IFN in response to infection, and that it
is essential for IFN-b expression (Honda et al., 2005).
Thus, IRF-7 is likely to be a component even of the early
enhanceosome.
We describe here the structure of a substantial part of
the IFN-b enhanceosome, including the PRDI and PRDII
regions in complex with the DNA-binding domains of
NFkB, IRF-7, and IRF-3. Together with our previously pub-
lished structure of the PRDIV-PRDIII half of the complex
(Panne et al., 2004) and with a structure of the overlapping
PRDIII-PRDI region also described here, we can construct
a complete picture of the DNA-proximal enhanceosome
architecture. Our structure shows IRF-3 bound to ISRE
sites A and C and IRF-7 to sites B and D in the PRDIII-I re-
gion of the enhancer, consistent with the dependence of
IFN-b transcription on both IRF family members. The com-
posite model shows that association of eight proteins with
enhancer DNA creates a continuous surface for the recog-
nition of 50 bp. Contrary to some suggestions, the DNA is
essentially straight. The various transcription-factor-bind-
ing sites overlap, and local conformational changes in the
DNA contribute to specificity and to transcription-factor
positioning, as already seen in the PRDIV-PRDIII structure
(Panne et al., 2004). The positive-regulatory domains of
the IFN-b enhancer form a single, composite binding ele-
ment. The unit of regulation for virus-activated transcrip-
tion is thus the entire nucleotide sequence rather than
individual PRD elements, consistent with strict conserva-
tion at almost every position of the enhancer in mamma-
lian genomes.
RESULTS
Strategy and Component Structures
of IFN-b Enhanceosome
We have previously reported a structure that contains the
ATF-2/c-Jun heterodimeric bZIP segments and two IRF-3
Figure 2. Structure of the NFkB:IRF-7:IRF-3:DNA Complex
Ribbons representation of the complex viewed (A) normal to and (B) along the DNA helical axis. The RHR of p50 is in light blue, and that of RelA in dark
blue. IRF-7D is in yellow, and IRF-3C in green. The two DNA strands are in gray. (C) Sequence of the DNA duplex corresponding to the PRDI-II region
of the IFN-b enhancer (85 to 51 nucleotides from the start site of transcription). (D) Protein-DNA contacts of IRF-3C. (E) Protein-DNA contacts of
IRF-7D.

DNA-binding domains, in complex with a DNA fragment
containing base pairs 102 to 72 (PRDIV and III) (Panne
et al., 2004). We now describe a crystal structure that
contains an IRF-3 DNA-binding domain, an IRF-7 DNA-
binding domain, and the Rel-homology regions (RHRs)
of NFkB p65 (RelA) and p50, bound to DNA base
pairs 85 to 51 (PRDI and PRDII) (Figure 2). Using a third
crystal structure that overlaps the first two, with four IRF-3
DNA-binding domains bound to a 57 bp enhancer DNA,
base pairs 102 to 46 (PRDIII and PRDI) (Figure S1),
we have reconstructed the complete set of DNA-binding
domains and enhancer DNA that determines the global
organization of the enhanceosome. The strategy is illus-
trated schematically in Figure 1. Before describing the new
structures in detail, we summarize necessary information
about the components from various other structures.
IRF DNA-Binding Domains
All IRF proteins have an N-terminal DNA-binding domain
of about 120 amino-acid residues, with a conserved a/b ar-
chitecture comprising a four-stranded antiparallel b sheet

Cell 129, 1111–1123, June 15, 2007 ª2007 Elsevier Inc. 1113
(b1–b4), three helices (a1–a3), and three long loops (L1–
L3) (Figures 2D and 2E) (Escalante et al., 1998, 2002a; Fujii
et al., 1999; Panne et al., 2004). Each DNA-binding domain
contacts the DNA backbone over a 12 bp stretch. A set of
protein-DNA contacts in the major and minor grooves
determines specificity for the IRF recognition sequence
50 -AANNGAAA-30 . Conserved residues Asn79, Arg81,
and Ser82 (IRF-3 numbering), all in a3, specify the down-
stream GAAA, and water-mediated contacts in the minor
groove from His40 determine the preferred upstream AA
(Figure 2D). Additional DNA contacts with nonconserved
residues Leu42, Arg78, and Arg86 explain the more re-
stricted binding specificity of IRF-3 (Panne et al., 2004).
Leu42, in L1, inserts into the minor groove adjacent to
His40 and contacts the base pair 50 to the consensus
site (50 -NAANNGAAA-30 ), disfavoring a G:C or C:G at
this position. (We have noted previously that this interfer-
ence at least partly explains the binding register at site A
[Panne et al., 2004].) Arg78 and Arg86 contact the regions
flanking the core GAAA repeat; although these side chains
are quite adaptable they favor a G:C or C:G on either side
of the core (Escalante et al., 2007; Panne et al., 2004).
IRF-7 is essential for viral induction of IFN-b (Honda
et al., 2005). We suggest, based on the specificities just
described, that sites B and D are selective for IRF-7, and
sites A and C for IRF-3. First, site D is suboptimal for
IRF-3 because of the potential interference between
Leu42 and G75 in the wild-type sequence. IRF-7 has ala-
nine instead of leucine at the corresponding position
(Figure 2E), allowing it to tolerate the guanine N2. Second,
sites A and C appear to prefer IRF-3 because they accom-
modate bidentate hydrogen bonding of Arg78 with two
successive guanines (Escalante et al., 2007). IRF-7 has
threonine at the corresponding position (Figure 2). Third,
the methyl group of this threonine can form a van der
Waals contact with the methyl group of a thymine just up-
stream of the consensus guanine, as in sites B and D.
Specificity for T at this position in IRF-7 sites and for G
in IRF-3 sites has also been detected in SELEX experi-
ments (Lin et al., 2000a), as well as in preferential IRF-3
and IRF-7 sites involved in transcription of the IFN-a genes
(Morin et al., 2002).
Alternation of the sites for IRF-3 and IRF-7 is also opti-
mal for concomitant binding by IRF dimers. IRF-3 is acti-
vated by phosphorylation of C-terminal residues, leading
to dimerization, nuclear translocation, and interaction with
CBP/p300 (Lin et al., 1998). Sites A and C are on the same
face of a DNA duplex, and the C termini of the DNA-bind-
ing domains at these sites project in the same direction,
about 40 Å apart. Binding of an IRF dimer at sites A and
C (or B and D) would allow attack from one side of the du-
plex and a simple geometry for the overall complex. The C
termini of the domains positioned at sites A and B project
in opposite directions, about 60 Å apart; binding of a dimer
at sites A and B (or C and D) would require wrapping of an
extended hinge around the enhancer. The N-terminal
DNA-binding domain and dimerization domains of IRF-3
are linked through a flexible segment of 70 amino-acid
1114 Cell 129, 1111–1123, June 15, 2007 ª2007 Elsevier Inc.
residues (Qin et al., 2005), long enough to accommodate
either arrangement, but the alternating characteristics of
the binding-site sequences clearly suggests that IRF-3
binds preferentially to sites A and C and IRF-7 to sites B
and D.
NFkB
The RHR of the NFkB heterodimer p50/RelA has been crys-
tallized on a number of different DNA sites (Berkowitz et al.,
2002; Chen et al., 1998a; Chen-Park et al., 2002; Escalante
et al., 2002b). In all these structures, the p50 subunit binds
at the 50 end and the RelA subunit at the 30 end of the kB site
(Figure 2). The main determinant of this orientation appears
to be a contact of His64 of p50 with the upstream guanine
(50 -GGGAAATTCC-30 ; in the description below, we number
the base pairs in this sequence 1–10); the homologous res-
idue in RelA is Ala, which does not confer specificity. There-
fore, the p50 half-site is generally described as 5 bp in
length (50 -GGGAAATTCC-30 ), and the RelA half site as 4
bp in length (50 -GGGAAATTCC-30 ) (Chen and Ghosh,
1999). The approximate dyad passes through the sixth
base pair, assigned to neither half site (Figures 2 and 3).
Structure of an NFkB:IRF-7:IRF-3:DNA
Complex at PRDI-II
Design of a Fusion Construct
IRF-7 has an insertion of 9 amino-acid residues between
helices a2 and a3 (Figure S3). With IRF-7 bound to site D,
this insertion projects toward loop L1 of p50. We consid-
ered that this loop might be involved in direct protein-
protein contacts with p50, but NFkB binds full-length
enhancer DNA together with the DNA-binding domains
of IRF-3 and/or IRF-7 without apparent cooperativity (Fig-
ure S2). Efforts to crystallize such complexes, either on
full-length enhancer or on a fragment containing sites C
and D plus PRDII, yielded only crystals with NFkB, IRF-
3, or IRF-7 bound alone to the DNA duplexes. To test
whether the specific locations of IRF-3 on site C and
IRF-7 on site D might be critical for cooperative assembly,
we designed polypeptide linkers between IRF-3 and IRF-7
to stabilize the relative order of the domains. We have
shown previously that adjacent IRFs can be linked in this
way, without affecting binding or structure (Panne et al.,
2004). Neither an IRF-3:IRF-3 or an IRF-3:IRF-7 dimer
showed cooperative binding with NFkB, however (Fig-
ure S2). From modeled structures based on IRF:DNA and
NFkB:DNA complexes, we noticed that the N and C ter-
mini of the domains are oriented favorably for construction
of the fusion protein RelA(RHR):IRF-7(DBD):IRF-3(DBD)
and of an extension of the same protein with an additional
IRF-7(DBD):IRF-3(DBD). (The colon [:] indicates a linker
sequence of varying length.) These fusion proteins were
coexpressed with the p50 RHR to generate an NFkB het-
erodimer with attached IRF domains. The purpose of the
fusion construct was to fix the relative order of individual
domains on the enhancer (IRF-3 on site C, IRF-7 on site
D, and NFkB on PRDII) and to stabilize the assembly.
Analysis of binding is shown in Figure S2. We experi-
mented with linkers of two lengths (see Experimental

Procedures); the shorter linkers allowed crystallization of
the heterodimer of p50 with RelA(RHR):IRF-7(DBD):IRF-
3(DBD) bound to a 35 base-pair DNA duplex spanning
PRDI and PRDII.
Structure Determination
The complex crystallized in space group P212121 (a = 95.5
Å, b = 116.4 Å, c = 134.8 Å) and gave measurable diffrac-
tion to dmin = 2.8 Å. We determined the structure by molec-
ular replacement and refined it to Rwork = 24.5%, Rfree =
28.6% (Table 1). The asymmetric unit contains one com-
plex; the 50 end of the DNA stacks against RelA of a symme-
try-related molecule, with the first two nucleotides of the
coding strand displaced from the duplex (Figures 2A and
2B). The 30 end does not have any crystal-packing interac-
tions. Thus, end-to-end DNA packing, as frequently found
in crystals of DNA:protein complexes, does not constrain
the DNA conformation. The refined model contains resi-
dues 19–291 of RelA, 37–350 of p50, 8–128 of IRF-7,
9–111 of IRF-3, and DNA nucleotide pairs spanning the
region from 85 to 51 of the enhancer (Figure 2). The de-
Cell 129, 1111–1123, June 15, 2007 ª2007 Elsevier Inc. 1115
Figure 3. Protein-DNA Contacts
Schematic diagram of protein-DNA contacts
generated with NUCPLOT (Luscombe et al.,
1997). Residues from ATF-2 are in red, c-Jun
in dark red, IRF-3A and C in green, and IRF-
7B and D in yellow. The RHR of p50 is in light
blue, and that of RelA in dark blue. The core
binding sites for each protein are indicated in
the corresponding colors. Blue lines indicate
hydrogen bonds, red lines, van der Waals
contacts.
signed linkers connecting the individual domains in the fu-
sion protein are disordered, as anticipated. The distances
spanned are 26 Å and 27 Å for the two linkers, much
less than the extended length of a 15-residue polypeptide
chain. The single-chain strategy is further validated by the
following observations. First, the conformations of individ-
ual domains of the fusion protein are identical to those
crystallized independently, in the context of other DNA
substrates and crystal lattices. Second, the DNA-binding
interfaces are just as expected for the individual constitu-
ents (see below). Contacts between the various DNA-bind-
ing domains and the DNA are summarized in Figure 3.
IRF-3 and IRF-7
Site C has one important deviation from the consensus
ISRE: the consensus GAAA is interrupted by a guanine
50 -TGAAAGGGAGAA-30 . There are two possible binding
registers for IRF-3. In one, a3 would lie over AGAA, pre-
serving the canonical two base-pair intersite spacing with
the downstream IRF-7; in the other, a3 lies over GAGA,
preserving a 2 bp intersite spacing with an upstream
Table 1. Data Collection and Refinement Statistics The principal difference between the IRF-7 DNA-binding
domain and those of IRF-1, IRF-2, IRF-3, and IRF-4, for
Data Collection NFkB:IRF-7:IRF-3:DNA
which structures have been determined previously (Esca-
Space group P212121 lante et al., 1998, 2002a; Fujii et al., 1999; Panne et al.,
Cell dimensions 2004), is in the three loops, L1–L3 (Figure S2). L2, between
helices a2 and a3, has a 9-residue insertion, which is in part
a, b, c (Å) 95.49, 116.37, 134.77
unstructured, reflecting a series of Gly and Pro residues.
Resolution (Å) 2.8 The DNA recognition helix, a3, has an N-terminal extension
Rsym 7.1 (60.0)a of 5 residues extending away from the DNA, so that it con-
tains 21 instead of the usual 16 residues (Figure 2E). Mod-
I / sI 37.6 (4.3)a
eling of IRF-1 onto the PRDI site proximal to NFkB showed
Completeness (%) 99 (100)a that loop L2 of either IRF would sterically overlap with loop
Redundancy 14.1 (13.5)a L1 of p50 (Escalante et al., 2002b). The insertion in IRF-7
leads to a rearranged L2, which together with a shift in L1
Refinement
of p50 (see below) avoids steric interference, allowing
Resolution (Å) 2.8 IRF-7 and NFkB to co-occupy the enhancer.
No. reflections 37592 Conserved interactions with GAAA in the major groove
of site D include hydrogen bonds between Arg96 (IRF-7
Rwork/Rfree 24.5/28.4
numbering) and the initial G and a nonpolar contact be-
No. atoms 7915 tween Ala98 and the methyl group of the thymine paired
Protein 6462 with the third A. The interactions of Cys97 with the two
central base pairs resemble those of its homolog, Ser82,
DNA 1428
in IRF-3 (Figures 2E and 3). The water-mediated, minor-
Water 25 groove contacts of His46 with two upstream adenines
Average B Factors (Å2) over All Residues in Each Chain (50 -AANNGAAA-30 ) are as described above for IRF-3, but
Ala 48, which replaces Leu42 of IRF-3, allows the protein
Protein 57.46, 66.92, 52.53, 107.51b
to accommodate an upstream G:C base pair. Also as de-
DNA 59.59, 49.46c scribed above, the van der Waals contact between Thr93
Water 36.08 and the C5-methyl group of T-71 strengthens the IRF-7
preference for sites B and D.
Rms Deviations
NFkB
Bond lengths (Å) 0.007 As in other NFkB-containing structures, including the
Bond angles ( ) 1.3 initially studied p50 homodimer:DNA complexes (Ghosh
a
Highest resolution shell is shown in parenthesis.
et al., 1995; Muller et al., 1995), homologous residues
b
RelA, p50, IRF-7D, IRF-3C. (two arginines and a glutamate) in both p50 and RelA rec-
c
Coding strand, Template strand. ognize the core guanine bases in each half-site. These
contacts, and others summarized in Figure 3, position
the two RHR-N domains with respect to the DNA duplex.
IRF-7 (not present in our crystals). We observe the latter We have compared our structure of the p50:RelA hetero-
configuration, which is also present in a structure of four dimer with others determined on a variety of DNA sites.
IRF-3 DNA-binding domains bound to PRDI-III (Escalante These structures differ among each other in the relative
et al., 2007). We can identify several structural reasons for orientations of the RHR-N and RHR-C domains, which
this selectivity. On the AGAA site, a minor-groove NH2 are flexibly hinged, and in the DNA conformations to which
from the first nucleotide of the G triplet (G79) would repel they accommodate. The dimeric RHR-C domains provide
His40. Binding at GAGA not only avoids this clash but also a convenient reference frame, as their contact is essen-
allows Arg78 to have the regularly observed bidentate tially invariant. Our structure differs from that of a p50:RelA
major-groove interaction with two consecutive guanines heterodimer bound with 12 bp of precisely the same DNA
(G78 and G77) (Figure 2D; also Escalante et al., 2007). Al- sequence (PRDII) (Berkowitz et al., 2002; Escalante et al.,
though Arg86 is disordered in our density map, it could 2002b) by as much as either of them differs from other
interact with the nonconsensus G in site C. In the context published structures (Figure S4 and Berkowitz et al.,
of the enhanceosome, the selection of this binding site 2002). That is, we can find in the comparison no clear cor-
permits a hydrogen bond to the main chain carbonyl of relation between DNA sequence and NFkB conformation.
Leu42 from Arg7 of the upstream IRF-3A, as observed in The DNA in our enhanceosome complex is longer than the
a four-site IRF-3:DNA structure described in the Supple- fragments used in earlier work with NFkB alone, and there
mental Data (Figure S1 and Table S1). IRF-7 does not are indeed additional phosphate backbone contacts,
have Arg at the corresponding position in its structure so made by S63, G65, and N136 of p50 and by S42, A43,
that the additional base pair between sites B and D does and G44 of RelA, that are not seen in the complex on a
not sacrifice this potential contact (Figure S3). 12 bp site. We believe that the comparison of our structure

1116 Cell 129, 1111–1123, June 15, 2007 ª2007 Elsevier Inc.
with the earlier PRDII complex (Berkowitz et al., 2002; Es-
calante et al., 2002b) calls into question the notion that
subtle differences in kB-site sequences can have reliable
‘‘allosteric’’ effects on the conformation of bound NFkB
(Chen-Park et al., 2002).
An interaction not present in earlier structures is the
contact between loops L1 of p50 and L2 of IRF-7 on site
D (Figure S4). The former shifts by about 5 Å toward
IRF-7, with respect to its position in the p50:RelA:PRDII
complex, but the interaction between the two loops is still
tenuous (Figure S4). The only evident contact is between
the side chain of Asn75 in p50 and the main-chain car-
bonyl of Gly68 in IRF-7. Loop L1 in p50 is relatively flexible,
and this single contact is unlikely to have any propagated
conformational effects on the rest of NFkB.
Model for the Complete Enhanceosome
The structure just described of NFkB:IRF-7:IRF-3 on PRDI-
II and the structure of ATF-2/c-Jun/IRF-3 on PRDIV-III to-
gether cover the entire IFN-b enhanceosome. Because
these structures overlap in the region spanning nucleo-
tides 72 to 85 of the enhancer, we could in principle
reconstruct the entire DNA-proximal assembly from these
two structures alone. We have chosen to use as an addi-
tional guide a structure containing four IRF-3 DNA-binding
domains that cover PRDIII–PRDI—that is, all four IRF sites.
That structure, which contained a single base-pair deletion
in PRDI, is described in the Supplemental Data (Figure S1
and Table S1). A related structure containing the wild-type
PRDI has been determined by another group (Escalante
et al., 2007), and the two are in excellent agreement. The
details of how the structure of NFkB:IRF-7:IRF-3 on
PRDI-II was overlapped with that of ATF-2/c-Jun/IRF-3
on PRDIV-III (Panne et al., 2004) are provided in Experi-
mental Procedures. Excellent spatial superpositions at the
ends of the overlapped structures validate our approach to
‘‘glue’’ together the PRDIV–PRDIII and PRDI–PRDII struc-
tures at the single interface between them (Figure S7). In
the model shown in Figure 4, we have replaced IRF-3 at
site B (from the structure of Panne et al., 2004) with IRF-
7, using criteria described in Experimental Procedures.
The fully assembled enhanceosome has a length of 160
Å. Binding of the eight proteins to DNA buries 13900 Å2
or 72% of the solvent-accessible surface area of the
enhancer DNA. We base the description below on the con-
catenated structure, although the analysis of local DNA
conformation is of course based on one or the other of
the individual coordinate sets.
Protein Interactions
A striking characteristic of the complex is the paucity of
specific protein interactions despite the close packing of
the various transcription-factor DNA-binding domains.
We observed this property in analyzing the ATF-2/c-Jun/
IRF-3/DNA complex, and it continues to be true all along
the length of the enhancer, as noted at various points in
the structure description above. Successive proteins do
contact each other but with relatively tenuous side-chain
interactions. Nonetheless, EMSA assays show that the
Cell 129, 1111–1123, June 15, 2007 ª2007 Elsevier Inc. 1117
IRF proteins bind cooperatively, with no detectable,
single-occupancy intermediate, provided that the DNA
substrate has at least two binding sites in a tandem orien-
tation (Escalante et al., 2007; Falvo et al., 2000; Fujii et al.,
1999; Panne et al., 2004). We therefore extend the con-
cept proposed earlier that some degree of cooperativity
can arise through DNA conformability in the absence of
strong protein contacts, when binding sites overlap (as
they do here) and the required or imposed DNA conforma-
tions at the overlapping sites are complementary (Panne
et al., 2004).
Enhancer DNA Conformation
The conformation of the 57 bp enhancer DNA exhibits local
variations about a straight, B form structure. The axis
traces a gently sinusoidal curve, with a net overall bend
of 13–15 and no sharp kinks (Figure 4A). Structures of
DNA-binding domains from IRF-1, IRF-2, IRF-3, and IRF-
4, complexed with DNA, show that these domains stabilize
a characteristic DNA conformation, in which the DNA du-
plex bends gently around the IRF recognition helix (a3) (Es-
calante et al., 1998, 2002a; Fujii et al., 1999; Panne et al.,
2004). This DNA conformation is present in both the IRF-
3 and IRF-7 sites and accounts for the sinusoidal path of
the helix axis. IRF-binding sites A, B, and C are separated
by 6 bp (just over half a turn of the DNA helix), and bends at
these sites are therefore of opposite phase. Site D is sep-
arated by 7 bp from site C, and because of this extra
base pair, the bends at sites C and D do not cancel each
other, leaving the enhancer with a 13–15 bend centered
on the A-tract in site D (Figure 4A). Similar conclusions fol-
low from the structure of four IRF-3 domains on PRDIII–
PRDI (Escalante et al., 2007). Thus, the net bend is largely
a consequence of the relative positions of the IRF domains,
as the ATF-2/c-Jun and NFkB sites in the complex are rel-
atively straight, consistent with measurements of bending
in solution (Falvo et al., 1995). The compression associated
with bending around each of the IRF domains leads to a pe-
riodic opening and closing of the major and minor grooves
along the enhancer (Figure S5). The closely spaced ar-
rangement of DNA-binding domains along the enhancer
would not allow major bends, even in response to con-
straints introduced by links between upstream and down-
stream elements outside the 57 bp segment in our crystals
(‘‘DNA looping’’), as neighboring proteins would then col-
lide (Figure 4B). The relatively undistorted conformation
of the enhancer rules out earlier suggestions that the DNA
might wrap around a transcription-factor core and contra-
dicts models that postulate long-range DNA bending as
a critical part of protein-DNA recognition at these sites
(Munshi et al., 1999).
HMGA1a Binding
The HMGA1a protein is thought to ‘‘orchestrate’’ assem-
bly and disassembly of the IFN-b enhanceosome, perhaps
through modification of DNA conformation and modula-
tion of protein-protein interactions (Yie et al., 1999). The
structure shows that an assembled enhanceosome can-
not accommodate HMGA1a, which binds in the DNA
minor groove through so-called ‘‘AT-hook’’ contacts from

one or more of three related, flexibly linked segments in
the short (110-residue) protein. There are four potential
HMGA1a sites in the IFN-b enhancer: the AT-rich region
around 60 in PRDII, a site 10 bp downstream around
position 48, and two sites flanking PRDIV around 100
and 88. HMGA1a variants form complexes with en-
hancer DNA, as detected by EMSA, but do not form stable
ternary complexes in the presence of the transcription
1118 Cell 129, 1111–1123, June 15, 2007 ª2007 Elsevier Inc.
Figure 4. Overall Structure of the IFN-
b Enhanceosome
(A) Side view of the complex, showing bending
of the DNA around the four IRF domains. The
red line shows the local DNA helical axis as
calculated with the program Curves (Lavery
and Sklenar, 1988). The RHR of p50 is in light
blue, and that of RelA in dark blue. IRF-7B
and D are in yellow, and IRF-3A and C in green.
ATF-2 is in light red, and c-Jun in dark red. The
two DNA strands are in gray.
(B) Molecular surface representation of the
IFN-b enhanceosome showing that the eight
polypetides form a composite surface for DNA
recognition. The two views, related by a 180
rotation as suggested by the arrow, are from
opposite sides of the complex.
factors. That is, complexes formed in the presence of
HMGA1a do not display further gel retardation when com-
pared to complexes formed in the absence of HMGA1a,
and there is no electron density for HMGA1a in maps
from cocrystallization efforts (Berkowitz et al., 2002;
Panne et al., 2004). One of the HMGA1a sites in PRDIV,
the minor groove of the AT-rich sequence at 88, is
blocked by loop L1 of IRF-7B (Panne et al., 2004), ruling
out two concomitant interactions at that end of the en-
hancer. At the other end, superposition of the NMR struc-
ture of the second and third DNA-binding segments of
HMGA1a bound to PRDII (Huth et al., 1997) onto the
NFkB-bound PRDII site in our structure shows that bind-
ing of NFkB and HMGA1a require very different minor-
groove widths. Thus, steric occlusion at PRDIV and con-
formational incompatibility at PRDII lead us to conclude
that HMGA1a is not part of the completed IFN-b enhan-
ceosome.
DISCUSSION
The virus-induced human IFN-b enhancer is arguably the
most thoroughly characterized transcriptional regulatory
element in any higher eukaryotic genome. The structure
reported here allows the assembly of a model of the full
enhancer bound to the DNA-binding domains of all the
relevant transcription factors. It shows in molecular detail
the interactions of ATF-2/c-Jun, IRF-3, IRF-7, and NFkB
with the enhancer DNA.
The nucleotide sequence of the IFN-b enhancer is
nearly invariant over roughly 100 million years of evolution,
unlike the sequence of the gene (Figure S6). Thus, the pre-
cise organization of the assembled transcription factors
has had strong and continuing selective advantage. More-
over, mutational analyses have shown that virtually every
nucleotide in the enhancer DNA sequence matters for
some aspect of the response to viral infection (Du and Ma-
niatis, 1992; Goodbourn and Maniatis, 1988; Thanos and
Maniatis, 1992). The enhanceosome structure accounts
for this conservation by showing that the transcription fac-
tors form a composite surface for recognition of the entire
sequence and that adjacent transcription-factor-binding
sites overlap (Figures 3 and 4). For example, IRF-3 and
IRF-7 specify additional bases around the core IRF-bind-
ing site 50 -AANNGAAA-30 through nonconserved amino-
acid residues such as Leu42, Arg78, and Arg86 in IRF-3
and Thr93 in IRF-7. These additional DNA contacts ex-
plain a number of observed sequence preferences (Lin
et al., 2000a; Morin et al., 2002). They also account for the
requirement of IRF-7 in the early IFN-b response to viral
infection (Honda et al., 2005) by showing how loop L2 of
IRF-7 at site D avoids interference with NFkB and how
DNA sequence just outside the cores of sites B and D
leads to a preference for IRF-7 over other family members.
A hallmark of combinatorial transcriptional control is
synergy, mediated largely in this case by enhanceosome
formation (Merika and Thanos, 2001; Struhl, 2001). Syn-
ergy implies strong cooperativity at some level of assem-
bly, such as direct interactions between adjacently bound
transcription factors. When c-Fos, c-Jun, and NFAT bind
the ARRE2 site of the IL-2 enhancer, contacts between
adjacent proteins do impart both cooperativity and spec-
ificity: an extended network of polar interactions, which
includes all three proteins and the DNA backbone, estab-
lishes a preferred orientation for the Fos:Jun heterodimer
on its binding site and a particular conformation for the
Cell 129, 1111–1123, June 15, 2007 ª2007 Elsevier Inc. 1119
two-domain RHR of NFAT (Chen et al., 1998b). Extended
contacts between transcription factors are noticeably ab-
sent in the IFN-b enhanceosome, however. Despite the
density with which the eight bound proteins are packed
along the essentially straight segment of enhancer DNA,
the structure and the binding measurements reported
here (Figure S2) and in our previous paper (Panne et al.,
2004) show that the relatively tenuous local protein inter-
faces between abutting DNA-binding domains impart
very little cooperativity. For example, the L2 loop of IRF-
7 has an insertion of 9 amino-acid residues (with respect
to IRF-3) between helices a2 and a3 (Figure S3). Although
this loop extends toward p50, IRF-7 does not bind coop-
eratively with NFkB to the enhancer (Figure S2), and the
structure shows that the glycine- and proline-rich L2
loop is largely disordered and moves out of the way to
accommodate loop L1 of p50 without making extensive
contacts (Figure S4).
What interactions can give rise to cooperativity of tran-
scription-factor association with the IFN-b enhancer in the
absence of strong contacts between adjacent proteins? In
principle, cooperative binding can arise through nucleo-
tide sequence-dependent structural changes in the DNA
that allow formation of complementary DNA conforma-
tions for adjacently bound transcription factors (Escalante
et al., 2002a; Klemm and Pabo, 1996; Panne et al., 2004).
This conformational complementarity appears to be the
case for ATF-2/c-Jun and all four IRFs (but not for NFkB,
which has a site that does not overlap that of its neighbor).
We have shown that cooperative binding of ATF-2/c-Jun
and IRF-3 depends on the inherent asymmetry of the
ATF-2/c-Jun-binding site and that modifying it into a con-
sensus AP-1 recognition element eliminates the coopera-
tivity (Panne et al., 2004). That is, the ATF-2/c-Jun site is
actually a composite element that accommodates not
just ATF-2/c-Jun but also part of the adjacent IRF-3. Sim-
ilarly, all four IRF-binding sites are composite elements,
and the structures show a remarkably precise sequence
organization to accommodate a specific array of IRFs.
Local complementarity of DNA conformation at overlap-
ping sites cannot, however, account for the strong in vivo
synergy of IFN-b gene regulation, as binding analyzed
by the EMSA experiments in Figure S2 would then have
shown a more strikingly cooperative character. Previous
work has shown that interactions beyond the DNA-bind-
ing domains provide additional driving force for coopera-
tive assembly. Have we failed to visualize important
pairwise interactions between the transcription factors?
Except for the dimerization domains of IRF-3 and IRF-7,
which not only hold the dimers together but also bind
the coactivator CBP/p300 (Qin et al., 2005), essentially
all of the regions of the various DNA-bound proteins
known to have well-defined, folded structure are included
in the structures and in our binding measurements. That is,
the remaining parts of ATF-2, c-Jun, and NFkB are prob-
ably flexibly extended, and various segments are known
to interact with specific coactivators or corepressors
or to serve as signals for nuclear localization or for
degradation. These extended regions are unlikely to form
specific contacts with each other or with the IRFs. The
absence of pairwise interactions in vitro, using purified
full-length activators, further supports this contention
(D.P., unpublished data).
The high-mobility group protein HMGA1a has also been
implicated in cooperative enhanceosome assembly (Tha-
nos et al., 1993). Unlike the stably folded, ‘‘architectural’’
HMG proteins such as LEF-1, HMG-1, and SRY, which al-
ter DNA conformation and create a platform for associa-
tion of transcriptional activators, HMGA1a merely requires
an accessible, AT-rich minor groove. The enhanceosome
structure shows that the mapped HMGA1a binding sites
are not accessible and that HMGA1a is unlikely to be part
of the final assembly. Enhanceosome assembly is asyn-
chronous (Munshi et al., 2001). HMGA1a could therefore
act as a molecular chaperone during different stages of
the assembly process and then dissociate from the final
complex—a mode of action also proposed for HMG-1 in
certain cases (Thomas, 2001).
Multivalent interactions of the coactivators, CBP and
p300, with all the assembled transcription factors partici-
pate in activating transcription directed by the IFN-b en-
hancer (Merika et al., 1998; Wathelet et al., 1998). CBP
and p300 are large, extended, flexible molecules with a se-
ries of domains, some widely spaced, that bind segments
of the activation regions of various transcription factors.
The IRF-binding domain (IBiD) near the C terminus of CBP
interacts with IRF-3 (Lin et al., 2001; Qin et al., 2005);
the KIX domain, near the N terminus, interacts with RelA
and c-Jun (Bannister et al., 1995); and the CH2 domain,
between KIX and IBiD, interacts with ATF-2 (Kawasaki
et al., 1998).
In transient transfection experiments with IFN-b reporter
genes, insertion of an integral DNA turn (10 bp) between
the PRDI–PRDII and the PRDIV–PRDIII domains of the
IFN-b enhancer does not compromise activation; insertion
of a half-integral turn of the helix (5 bp) between the sites
essentially disables the enhancer (Thanos and Maniatis,
1995). These experiments reveal the importance of the po-
sition of transcription factors on the face of the DNA helix
in the assembly of the preinitiation complex, and they illus-
trate the adaptability of CBP and p300 in spanning vari-
able intervals between DNA-bound transcription factors.
They do not, however, reflect all the biological specificity
that has led to the evolutionary invariance of the enhancer
sequence. In particular, the transfection experiments are
unlikely to reflect the subtleties of enhanceosome assem-
bly and enhancer function of the endogenous gene in the
context of chromatin. For example, the level of induction
of the endogenous gene is orders of magnitude higher
than that observed with the transfected reporter (T.M. un-
published data), and thus the effects of insertions on
cooperative binding might not be observed at all in the
transfection experiments.
The IFN-b enhanceosome is a precise and specific as-
sembly of ‘‘generic’’ transcription factors that participate
in many other regulatory complexes as well. Faithful coin-
1120 Cell 129, 1111–1123, June 15, 2007 ª2007 Elsevier Inc.
cidence detection requires that a functional response
should occur only when the right set of transcription fac-
tors is on the enhancer and only when all those factors
are indeed present. The structure shows that this combi-
natorial specificity is encoded not just in the various bind-
ing sites but also in their overlap and in their positions with
respect to each other. That is, precision of the assembly
contributes directly to its specificity (e.g., to the require-
ment for IRF-3 and IRF-7), even in the absence of extended
protein-protein interfaces. The strict evolutionary conser-
vation of the IFN-b enhancer sequence correlates with its
organizational precision, and we suggest that other strictly
conserved enhancer sequences—for example, the 300 bp
IL-2 enhancer/promoter—may have similar structural
characteristics. These characteristics also imply that non-
consensus-binding-site sequences can have critical func-
tional importance, a property that will need to be included
in computational algorithms for detecting transcription-
factor sites in genome sequences.
The IFN-b enhanceosome structure further shows that
cooperativity of assembly probably resides at a level of
interaction not represented by contacts between neigh-
boring DNA-binding domains but probably at the level of
coactivators. The flexibility of CBP/p300 allows it to serve
as a signal integrator not only for enhanceosomes of
tightly defined geometry but also for ‘‘modular’’ enhancers
with more variably spaced binding sites. One of the best-
studied modular elements, the even-skipped stripe-2 en-
hancer of Drosophila (Small et al., 1991), shows local
evolutionary conservation over segments longer than
a single transcription-factor-binding site, even when the
larger-scale organization of the enhancer is clearly vari-
able. Thus, conserved subelements may have a precise,
enhanceosome-like molecular architecture within a gener-
ally more flexible complete enhancer (Ludwig et al., 2000,
2005). A generic adaptor (CBP/p300) would then pass on
to the Pol II machinery a summary of tightly regulated
signals from several specifically arrayed sets of generic
activators.
EXPERIMENTAL PROCEDURES
Protein Constructs and Structure Determination
To obtain a defined complex for crystallization, we created a single-
chain construct linking residues 19–291 of RelA, 8–128 of IRF-7, and
4–113 of IRF-3, as well as a variant construct with an additional IRF-
7/IRF-3 pair at its C terminus. The domains were joined by flexible
linkers, L1–L4. In previous work, we joined two IRF-3 DNA-binding do-
mains in tandem with a 26-residue flexible linker (Panne et al., 2004)
and used this covalently linked dimer, which bound the enhancer
more tightly than a pair of unlinked domains, for crystallization (Panne
et al., 2004). In that work, we also crystallized the components in the
absence of the covalent linker and found no structural differences.
Thus the linker allowed stabilization of the assembly without introduc-
ing structural constraints. Moreover, the structure reported here of four
IRF-3 DNA-binding domains bound to the INF-b enhancer was ob-
tained using no linker between individual IRF-3 domains (Figure S1),
and it agrees very well with the linked complexes in regions of overlap
(see Results). We are therefore confident that the linkers do not perturb
the structures of the DNA:protein complexes.
 Coding sequences for the RelA-IRF-7-IRF-3 fusion proteins used
here were cloned with a C-terminal hexahistidine-tag into the pET
Duet vector (Novagen), along with the RHR (residues 37–350) of p50
as the second encoded chain. Linker design was initially based on
the one used previously (Panne et al., 2004). These constructs failed
to yield crystals, however, and we then further adjusted the linker se-
quences. The most important change was to reduce the linker length
in L2 and L4 (linking IRF-7 to IRF-3) from 54 to 16 amino acids. We
used the restriction enzyme SapI for cleavage at the fusion junctions,
allowing us to design and clone the linker sequences without introduc-
ing additional, unwanted amino acids. The NFkB:IRF-7:IRF-3 con-
struct with the shorter linker sequences yielded the crystals reported
here. Expression, purification, crystallization, and data collection
were performed as described in Supplemental Data.
The structure was determined by molecular replacement using
MOLREP (Vagin and Teplyakov, 1997). As search models, we used the
structures of NFkB bound to DNA from the PRDII site of the INF-b en-
hancer (1LE5; Berkowitz et al., 2002) and of the IRF-3:DNA complex
(1T2K; Panne et al., 2004). After locating the NFkB:DNA complex, we
fixed its position and orientation and searched for the two IRF:DNA
complexes. The additional DNA was built by placing the phosphates
of nucleotide pairs visually into density and restraining their positions
during initial refinement. The planarity of the base pairs and the sugar
pucker were restrained to conform to standard B-DNA. The dihedral
torsion angles of a helices were restrained to those of an a-helical con-
formation. In later refinement cycles, these conformational restraints
were removed. Iterative model building was performed using the
programs O and Coot (Emsley and Cowtan, 2004; Jones et al.,
1991); refinement calculations used CNS 1.2 (Brunger et al., 1998).
The DNA helical parameters were analyzed with 3DNA (Lu and Olson,
2003), and the global helical axis was plotted with Curves (Lavery and
Sklenar, 1988).
Model for the Complete Enhanceosome
The model in Figure 4 was obtained by superposing the overlapping
parts of two structures, those of ATF-2/c-Jun/IRF-3/DNA (1T2K) and
NFkB:IRF-7:IRF-3:DNA (2O6I), with the structure of four IRF-3 DNA-
binding domains bound to the full-length enhancer (2O6G) as a guide.
Ca residues 5–110 from IRF-3B (chain B, 1T2K) were superposed, with
the program O, with those of IRF-3B (chain F, 2O6G). These two chains
superpose with a root mean square deviation (rmsd) of 0.54 Å. Ca res-
idues 9–110 from IRF-3C (chain D, 2O6I) were superposed with those
of IRF-3C (chain G, 2O6G). These two chains superpose with a rmsd
of 0.98 Å. The two structures contain overlapping DNA nucleotides
(region 85 to 72 of the enhancer), one set of which was removed in
the final assembly. The excellent superposition in this region can be
seen in Figure S7 (rmsd 1.2 Å). Finally, to complete the model, Ca res-
idues 6–65 and 90–127 of a copy of IRF-7D (chain C, 1O6I) were super-
posed with residues 6–65 and 72–109 of IRF-3 (chain B, 1T2K). These
two chains superpose with a rmsd of 0.98 Å as shown in Figure S7.
Supplemental Data
Supplemental Data include Experimental Procedures, seven figures,
one table, and a coordinate file (pdf format) for the composite model
in Figure 4 and can be found with this article online at http://www.
cell.com/cgi/content/full/129/6/1111/DC1/.
ACKNOWLEDGMENTS
We thank Carlos Escalante and Aneel Aggarwal for communicating
results prior to publication and Michael Carey and Ernest Fraenkel
for comments on the manuscript. Data were collected at beamline
8.2.1 at the Advanced Light Source, Lawrence Berkeley Laboratory,
and at BioCARS beamline 14C at the Advanced Photon Source,
Argonne National Laboratory. S.C.H. is an Investigator in the Howard
Hughes Medical Institute. T.M. was supported by grant R01AI020642
from the National Institutes of Health.
Cell 129, 1111–1123, June 15, 2007 ª2007 Elsevier Inc. 1121
Received: December 20, 2006
Revised: April 26, 2007
Accepted: May 11, 2007
Published: June 14, 2007
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Accession Numbers
Coordinates and structure factors have been deposited in the RSCB
Protein Data Bank with accession codes 2O6G for the IRF-3/DNA
structure and 2O6I for the NFkB:IRF-7:IRF-3:DNA structure.